The goals of this proposal are to examine cellular mechanisms which regulate Na+ transport across renal tissue. The two tissues which will be used are a mouse cortical collecting tubule cell line, MC-1, and an amphibian distal nephron line, A6. Both of these tissues have trans- epithelial sodium transport which can be induced by the hormone, aldosterone, and blocked by the diuretic, amiloride. The primary emphasis of this proposal will be an examination of apical Na+ channels using patch- voltage clamp and biochemical methods. The rationale for the experiments is that the patch clamp method allows characterization of single transport proteins uncomplicated by interaction with other cellular processes. Also, patch-clamp allows access to the inner surface of the cell membrane which is in general inaccessible in intact tissue. Such accessibility allows an examination of intracellular factors which may regulate Na channels. The specific aims of the project fall into two major categories. The first is to examine the mechanisms for regulation of renal sodium channels with particular emphasis on regulation which involves (1) apical-membrane- associated proteins like G-proteins and lipases, (2) phosphorylation and dephosphorylation, (3) steroid hormones, and (4) cellular cytoskeletal elements. The second major direction will be to examine the ability of these regulatory cascades to alter sodium channel activity in a physiological context. Using biochemical methods, we will examine phospholipase, kinase, and methylase activity in response to putative physiological stimuli including cell volume, eicosanoids, vasopressin, aldosterone, and insulin. the primary hypothesis which motivates this project is that there are three hierarchical levels of Na channel regulation: first, apical membrane-delimited regulatory elements which directly control Na channels; second, cellular second messenger systems which interact with the apical regulatory elements; and, third, systemic hormonal agents and their cellular receptors which activate the first two levels of regulation. Progress during the preceding grant period includes a demonstration (1) of at least four types of amiloride-blockable channels in A6 cells or CCT principal cells, (2) that anti-diuretic hormone alters sodium transport by changing the density of sodium channels with no change in open probability, (3) that aldosterone alters sodium transport primarily by changing the open probability, not by changing channel density, (4) that the mechanism of aldosterone's increase in sodium transport involves membrane protein methylation, (5) G proteins modulate sodium transport by activating phospholipase C/protein kinase C, and (6) PKC reduces channel open probability.